Chronic feeding of a choline-deficient-L-amino acid-defined (CDAA) diet containing no carcinogens exerts a strong hepatocarcinogenicity in rats through the development of apparently preneoplastic, focal lesions in the background presence of repeating hepatocyte death and regeneration as well as fibrosis. Oxidative stress appears to play major roles in its underlying mechanisms in association with alteration on the status of various signaling molecules. Phenyl N-tert-butyl nitrone (PBN), a radical trapper, has been shown to inhibit the development of preneoplastic lesions in the early phase of this dietary hepatocarcinogenesis by apparently inhibiting oxidative stress, inducible cyclo-oxygenase activity and fibrogenesis (Floyd et al., 1998).
Reactive oxygen species (ROS) have been implicated in cancer development for many years. A prime example where ROS are strongly implicated is the model system where feeding a choline deficiency (CD) diet to rats leads to hepatocellular carcinoma (HCC) development, i.e. in the complete absence of exposure to any exogenous known carcinogen. Utilizing this model, the present invention concerns novel observations that make it possible to link ROS with key signal transduction pathways that have been shown to be fundamental in cancer initiation and development. The present inventors have shown that mitochondria from CD-livers are changed such that they mediate a significantly higher yield of H2O2 production. Additionally, for the first time the present inventors have shown that PBN (xcex1-phenyl-tert-butyl nitrone) and its derivatives are nitrone-based free radical traps and, significantly reduce preneoplastic nodule development as well as inhibit hepatocellular carcinoma (HCC) formation at very low levels of the compound. PBN and the like are the most potent anti-carcinogens ever studied in this model. To understand these observations the inventors postulate that the CD-regimen mediates changes in mitochondrial membranes such that they produce enhanced levels of H2O2 and that PBN and the like significantly inhibit the excess H2O2 production by acting at Complex I. The present inventors further postulate that excess H2O2 causes an enhanced inactivation of the PTEN tumor suppressor protein, which causes a loss of its phosphatase activity and thereby mediates a shift toward the activation of the AKT-kinase pathway resulting in a decrease in apoptosis-mediated processes but an increase in oncogenic events. The inventors also propose that the cells in preneoplastic nodules which develop in CD-livers are predisposed toward ontogenesis (as opposed to apoptosis) because of the action of excess H2O2 and certain growth factors (most likely TGFxcex21) and that PBN and the like alter these processes through both inhibition of excess H2O2 production and also by suppression of enhanced signal transduction processes. The inventors believe that PBN and the like act to cause preneoplastic nodule cells to become predisposed toward apoptic processes leading to inhibition of tumor development.
Studies on the Pharmacological Action of PBN
The compound PBN was first synthesized in the 1950""s, but in 1968 it was discovered to be very useful to trap and stabilize free radicals in chemical reactions and hence it was termed a spin-trap (Janzen 1971). Although PBN is the prototype spin-trap several other nitrones have been synthesized and found useful to trap and characterize free radicals in chemical reactions. These spin traps were used in chemical reactions first, but in the mid-1970""s they began to be used to trap free radicals in biochemical and biological systems (Floyd et al. 1978; and Poyer et al. 1978, for example). Pharmacokinetic studies have shown that PBN is readily and rapidly distributed almost equally to all tissues, has a half-life in rats of about 132 minutes and is eliminated mostly in the urine. Relatively few metabolism studies have been done, but it is known that some ring hydroxylation (primarily in the para position) of the compound occurs in the liver. Novelli first showed that PBN could be used to protect experimental animals from septic shock (Novelli et al. 1986), and indeed this was later confirmed by other groups (Pogrebniak et al. 1992). The use of PBN and derivations as pharmacological agents began after discoveries in 1988 that showed that PBN had neuroprotective activity in experimental brain stroke models (Floyd 1990; Floyd et al. 1996; and Carney et al. 1991). These results were repeated and extended, (i.e. see References Clough et al. 1991; Cao et al. 1994; Folbergrovaet al. 1995; Pahlmark et al. 1996, for example). The present inventors have summarized the extensive neuroprotective pharmacological research effort on PBN and derivatives (Floyd 1997; Hensley et al. 1996). In addition to neurodegenerative diseases, PBN has been shown to protect in other pathological conditions where ROS-mediated processes are involved, including diabetes and many other conditions. The mechanistic basis of why PBN and some of its derivatives are so neuroprotective in experimental stroke and several other neurodegenerative models has not been completely elucidated yet. However, it is clear that its action cannot simply be explained by its ability to trap free radicals. In fact the present inventors"" research effort on the mechanistic basis of PBN""s action now shows that it is acting by suppressing gene induction (Floyd 1997; Hensley et al. 1996; Miyajima et al. 1995; Tabatabaie et al. 1996; and Hensley et al. 1997), most likely by acting on oxidation-sensitive signal transduction processes (Robinson et al. 1999). In fact PBN seems to be acting by suppressing signal transduction enhanced ROS formation by mitochondria (Hensley et al. 1998). These findings and ideas have arisen from the study of neurodegenerative processes. It should be emphasized, however, that PBNs action in preventing CD carcinogenesis may be different than those found in the neurodegenerative disease models. A specific mechanism of action does not limit the present invention.
PBN is Protective in Choline-deficiency Model
Earlier studies showed that PBN administered in drinking water was very protective in the CD-model. The results were assessed after 12 weeks on the regime (Nakae et al. 1998). The research brought out several important points (1) PBN, even at the lowest level, drastically reduced the size of neoplastic nodules (from 1.92 mm3 in CDAA only to 0.33, 0.17 and 0.10 mm3 for the CDAA plus PBN treated at 6, 30 and 60 mg/kg-day respectively, see Table 1 of Nakae et al. 1998).
There was less effect of PBN on nodule number, i.e. 190 per mm3 for CDAA only to 170, 149 and 142 for the 6, 30 and 60 mg/kg- day respectively, (see Table 1 of Nakae et al. 1998). (2) PBN significantly reduced connective tissue proliferation. (3) Increasing concentrations of PBN reduced 8-OHdG content (a marker of DNA oxidation) in the CD-livers. (4) PBN reduced the amount of PGE2 in the CD-livers by about 50% at the highest dose but it had no effect on COX-II expression, either the mRNA or protein level. In summary then the fact that the very lowest level of PBN decreased the nodule size by 83% but only decreased the nodule number by 11% indicates to us that nodule size is the most sensitive parameter to PBN treatment. There was some effect on PGE2 levels but only at the highest levels of PBN and this probably had to do with it acting as a catalytic inhibitor of the enzyme per se.
To highlight the potency of PBN relative to other chemicals that have been tested in the CDAA model, it is instructive to compare results, which were obtained by the Nakae-Konishi group (see Mizumoto et al. 1994; Endoh et al 1996; and Nakae 1999). The data clearly show that PBN is the most effective compound tested in the CDAA regimen in reducing the size of the preneoplastic nodules and in preventing an increase in the 8-OHdG content. The effectiveness of PBN on nodule size is much more potent than comparable amounts of the other inhibitors, most of which are free radical scavengers. The only other compounds that seemed to have some effect, albeit at higher levels, were nordihydroguaiaric acid (NGDA) and CV3611. CV3611 is the fatty acid ester of ascorbate. NGDA at the 0.1% level lowered nodule size, by 39% and CV3611 at the 0.05% level caused a 44% lowering. In contrast, PBN at the lowest level amount given (6 mg/kg) decreased nodule size by 83%; and by 95% at the highest level. NGDA was tested because of its known inhibition of lipoxygenase activity, but it has also been recently shown to antagonize tyrosine kinases (Hensley 1998). BPB (p-bromophenocybromide) was used as an inhibitor of phospholipase A2 activity but as the data show, this compound had little activity in suppressing the size of the nodules (Endoh 1996). Acetylsalicylic acid had some effect (but not nearly as potent as PBN) on nodule size and nodule number, as well as 8-OHdG content (Endoh 1999). Alpha tocopherol and ascorbate as well as trolox were studied by they had very little effect on any of the parameters (Mizumoto et al. 1994). It should also be noted that none, if any, of the inhibitors have any effect on fatty liver development. The wide variability in effectiveness of various antioxidants in this model and their lack of effect on fatty liver development seems to be a consistent finding. A striking case in point involves antioxidant effectiveness of compounds inhibiting lipid peroxidation in rat liver mocrosomes versus their action in the CDAA model. Data collected by Janzen et al. 1994 demonstrate that Trolox and BHT show quite striking activity in ability to inhibit rat liver microsome peroxidation (IC50 of 40xcexcM and 6xcexcM respectively) whereas PBN is about one thousand-fold less effective (IC50 =5 mM). Yet PBN is very effective in the CDAA model (Nakae et al. 1998) but BHT and Trolox are not (Ghoshal et al. 1990; Mizumoto et al. 1994). This comparison amply illustrates the point that the action of inhibitors in the CDAA model cannot simply be explained by their antioxidant or radical scavenging properties alone.
While earlier studies have indicated a possible connection between the occurrence of preneoplastic nodular lesions and the presence or absence of PBN in rats on a CDAA diet, those of skill in the art understand that these preneoplastic nodular lesions are not dependently predictable of frank cancer development. The present invention establishes that nitrone reductants such as PBN as its derivatives are in fact effective in inhibiting the development of actual cancerous lesions. Those of skill in the art will understand that this discovery has great implications for one of the major health problems of our day.
The present invention involves a method for inhibiting initiation or development of cancer or tumor development. The method comprises enterally administering an effective dose of a nitrone free radical trapping agent. The administering is preferably enteral by supplementing food or drink.
Any phenyl, alkyl-substituted nitrone is preferred in the practice of the present invention. This is a narrow definition because the basic core structure of the phenyl nitrone is so simple that there are only a few thousand practically synthesized derivatives. A preferred nitrone is an aryl N-alkyl nitrone. The alkyl is tertiary (tert) butyl although other alkyls, cycloalkyls and the like may be used. Preferred aryls are phenyl, 3-hydroxyphenyl and 4-hydroxyphenyl and the like. Both new aryls and alkyls may be used once one of skill in the art performs tests as described herein to identify effective nitrones, find optimal doses, delivery and timing schedules. Dietarily administering an effective dose of a nitrone free radical trapping agent to a subject is a preferred administrative route although other routes may be found effective in particular situations.
The present invention more preferably involves a method comprising enterally administering an effective dose of 3-hydroxyphenyl N-tert-butylnitrone or 4-hydroxyphenyl N-tert-butylnitrone to prevent or inhibit cancer. In most cases an effective dose is from about 0.5 to about 60 mg/kg body wt. per day. In one preferred embodiment, the present invention involves a method for inhibiting hepatocarcinogenesis, the method comprising dietarily administering to a subject an effective dose of at least one of phenyl N-tert-butylnitrone, 3-hydroxyphenyl N-tert-butylnitone or 4-hydroxyphenyl N-tert-butylnitrone. Subjects to be treated include those with a family history of cancer such as prostate, breast, liver or other cancer, as well as subjects believed to have been exposed to a carcinogenic environment such as excess UV irradiation, radiation, food contaminated with carcinogens, etc. In cases where the dietary administration is through supplementation of a food component, the nitrone content effective amount may be from about 0.005 w/w % to about 0.1 w/w % of the diet being administered.
Other tumor models where PBN and its chemical derivatives are likely to be active include HCC development caused by infection from hepatitis B virus and hepatitis C virus. Many tumors that afflict humans progress through a preneoplastic nodular stage and evidence indicates that ROS or conditions that exacerbate ROS formation are important in tumor development. Therefore, we consider it likely that the development of many human tumors may be held in check by daily administering of PBN or one of its effective chemical derivatives at very low levels (perhaps at 1 mg or less per day).
The present invention also involves a nitrone free radical trapping agent for use in the preparation of an anti-carcinogenic diet and the preparation of such supplemented diets. Again an aryl N-alkyl nitrone free radical trapping agent is preferred for use in the preparation of an anti-carcinogenic diet. The most preferred nitrones are 3-hydroxyphenyl N-tert-butylnitone and 4-hydroxyphenyl N-tert-butylnitrone, individually or in combination for use in the preparation of an anti-carcinogenic diet.
While earlier studies have indicated a possible connection between the occurrence of preneoplastic nodular lesions and the presence or absence of PBN in rats on a CDAA diet, those of skill in the art understand that these preneoplastic nodular lesions are not dependently predictable of frank cancer development. The present invention establishes that nitrone reductants such as PBN as its derivatives are in fact effective in inhibiting the development of actual cancerous lesions. Those of skill in the art will understand that this discovery has great implications for one of the major health problems of our day.
Examples where PBN (or its potent derivatives) are expected to be active in preventing frank tumor development included in addition to those in the liver, those that develop in most organs including stomach, colon, breast, pancreas, prostate, skin, head and neck, as well as the blood stream.
The present invention also involves a nitrone free radical trapping agent for use in the preparation of an anti-carcinogenic diet and the preparation of such supplemented diets. Again an aryl N-alkyl nitrone free radical trapping agent is preferred for use in the preparation of an anti-carcinogenic diet. More preferred nitrones are 3-hydroxyphenyl N-tert-butylnitone, 2-hydroxyphenyl N-tert-butylnitone, 2-sulfoxyphenyl N-tert-butylnitone and 4-hydroxyphenyl N-tert-butylnitrone, individually or in combination for use in the preparation of an anti-carcinogenic diet. Those subjects most likely to beneficially receive the nitrones of the present invention would include; 1) those having had pretumor tests indicating a high probability of the presence of tumors, 2) those exposed to very potent carcinogenic environments and their probability of tumor progression is high, and 3)to those whose genetic predisposition makes their likelyhood of tumor development high in the tumors that the particular nitrone will be effective in controlling